Inhibition of IL-17 production

ABSTRACT

The invention concerns inhibition of the production of proinflammatory cytokine interleukin-17 (IL-17) by T cells, using an antagonist of interleukin-23 (IL-23). The invention further concerns the use of IL-23 antagonists in the treatment of inflammatory diseases characterized by the presence of elevated levels of IL-17. IL-23 antagonists include, without limitation, antibodies specifically binding to a subunit or IL-17 or an IL-17 receptor. The invention additionally concerns induction of IL-7 production by using an IL-23 agonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a non-provisional application filed under 37 C.F.R.1.53(b), claiming priority under U.S.C. 119(e) to provisionalApplication Serial No. 60/423,090 filed Oct. 30, 2002.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention concerns inhibition of the production ofproinflammatory cytokine interleukin-17 (IL-17) by T cells, using anantagonist of interleukin-23 (IL-23). The invention further concerns theuse of IL-23 antagonists in the treatment of inflammatory diseasescharacterized by the presence of elevated levels of IL-17.

[0004] 2. Description of the Related Art

[0005] IL-17 is a T cell derived pro-inflammatory molecule thatstimulates epithelial, endothelial and fibroblastic cells to produceother inflammatory cytokines and chemokines including IL-6, IL-8, G-CSF,and MCP-1 (S. Aggarwal, A. L. Gurney, J Leukoc Biol 71, 1 (2002); Z. Yaoet al., Immunity 3, 811 (1995); J. Kennedy et al., J Interferon CytokineRes 16, 611 (1996); F. Fossiez et al., J Exp Med 183, 2593 (1996); A.Linden, H. Hoshino, M. Laan, Eur Respir J 15, 973 (2000); X. Y. Cai, C.P. Gommoll, Jr., L. Justice, S. K. Narula, J. S. Fine, Immunol Lett 62,51 (1998); D. V. Jovanovic et al., J Immunol 160, 3513 (1998); and M.Laan et al., J Immunol 162, 2347 (1999)).

[0006]IL-17 also synergizes with other cytokines including TNF-α andIL-1β to further induce chemokine expression (Jovanovic et al., supra,and M. Chabaud, F. Fossiez, J. L. Taupin, P. Miossec, J Immunol 161, 409(1998)). Levels of IL-17 are found to be significantly increased inrheumatoid arthritis (RA) synovium (S. Kotake et al., J Clin Invest 103,1345 (1999); and M. Chabaud et al., Arthritis Rheum 42, 963 (1999)),during allograft rejection (M. A. Antonysamy et al., Transplant Proc 31(1999); M. A. Antonysamy et al., J Immunol 162, 577 (1999); C. C. Loong,C. Y. Lin, W. Y. Lui, Transplant Proc 32 (2000); and H. G. Hsieh, C. C.Loong, W. Y. Lui, A. Chen, C. Y. Lin, Transpl Int 14, 287 (2001)), andin other chronic inflammatory diseases including multiple sclerosis (K.Kurasawa et al., Arthritis Rheum 43, 2455 (2000)) and psoriasis (C.Albanesi et al., J Invest Dermatol 115, 81 (2000), and B. Homey et al.,J Immunol 164, 6621 (2000)). Although clearly produced by activated Tcells, previous reports have not provided clear classification of IL-17within the paradigm of Th1 and Th2 polarized cytokine profiles.

[0007]IL-23 is a heterodimeric cytokine, sharing a subunit, termed p40,with interleukin-12 (IL-12), that combines with a unique subunit, p19(B. Oppmann et al., Immunity 13, 715 (2000)). IL-23 has been reported topromote the proliferation of T cells, in particular memory T cells (D.M. Frucht, Sci STKE 2002 Jan. 8; 2002(114):PE1). Transgenic p19 micehave been recently described to display profound systemic inflammationand neutrophilia (M. T. Wiekowski et al., J Immunol 166, 7563 (2001)).

[0008] No correlation has so far been established between the expressionand biological roles of the IL-17 and IL-23 cytokines.

SUMMARY OF THE INVENTION

[0009] In one aspect, the invention concerns a method for inhibitinginterleukin-17 (IL-17) production by T cells comprising treating the Tcells with an antagonist of interleukin-23 (IL-23).

[0010] In another aspect, the invention concerns a method for thetreatment of an inflammatory disease characterized by elevatedexpression of interleukin 17 (IL-17) in a mammalian subject, comprisingadministering to the subject an effective amount of an antagonist ofinterleukin-23 (IL-23).

[0011] In yet another aspect, the invention concerns a method foridentifying an anti-inflammatory agent comprising the steps of:

[0012] (a) incubating a culture of T cells with IL-23, in the presenceand absence of a candidate molecule;

[0013] (b) monitoring the level of IL-17 in the culture; and

[0014] (c) identifying the candidate molecule as an anti-inflammatoryagent if the level of IL-17 is lower in the presence than in the absenceof such candidate molecule.

[0015] In a further aspect, the invention concerns a method for inducingIL-17 production in a mammalian subject comprising administering to saidsubject an IL-23 agonist.

[0016] In all aspects, the antagonist or agonist preferably is ananti-IL-23 or anti-IL-23 receptor antibody, including antibodyfragments. The inflammatory disease preferably is a chronic inflammatorycondition, such as, for example, rheumatoid arthritis (RA), graft versushost reaction that may lead to allograft rejection, multiple sclerosis(MS) or psoriasis. The induction of IL-17 production is typically usefulin patients subjected to bacterial infection, such as, for example,infection Mycobacterium tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1: IL-17 production in different cell types (A): Single cellsuspensions of spleen were prepared from C57/BL-6 mice and mononuclearcells were isolated from suspended splenocytes by density gradientcentrifugation. 2×10⁶ cells/ml were cultured in the presence or absenceof microbial lipopeptide LBP (100 ng/ml), LPS (100 ng/ml) or LTA (100ng/ml) for 3 days, following which the cells were collected and analyzedfor IL-17 using ELISA. (B): Purified T cells were obtained from murinesplenocytes following positive selection of FACS sorted CD90 labeledcells. These cells were cultured (1×106 cells/ml) in presence or absenceof plate-bound anti-CD3 (5 μg/ml), or supernatant from activateddendritic cells (LPS-treated) for 3 days and culture supernatantscollected and analyzed for IL-17 levels using ELISA kit. Dendritic cellswere derived from macrophages (obtained as adherent population fromsplenocyte suspension), by treating macrophages with rmGM-CSF (2 ng/ml)and rmIL-4 (1000 U/ml) for 4 days, washing and re-activating using LPS(0.5 μg/ml). Representative results from 3 independent experiments areshown.

[0018]FIG. 2: IL-23 stimulates production of IL-17 A. Mononuclear cellsisolated from splenocytes were cultured (2×10⁶ cells/ml) with 100 U/mlrecombinant IL-2 and were incubated in presence or absence of variousconcentrations of IL-23 (0.1-1000 ng/ml) for 6 days. Levels of IL-17accumulated in culture supernatants were measured using ELISA. B.Changes in mRNA levels for IL-17 in response to IL-23 treatment weremeasured by quantitative RT-PCR. Plotted is the relative change in Ct(cycle threshold) of the PCR reaction. Data for each sample isnormalized to the glyceraldehyde-3-phosphate dehydrogenase mRNA levelpresent in each sample and then normalized again between samples to thelevel of IL-17 mRNA present in the time zero unstimulated conditions. Aseach Ct corresponds to a PCR cycle, one Ct is approximately equal to a2-fold change in mRNA abundance. The approximate mRNA fold differencefor 5 Ct and 10 Ct changes are indicated in parenthesis. The experimentwas performed with splenocytes from 4 mice, and the individual datapoints are represented with x and the average Ct change is indicated bybar columns. C. Changes in mRNA levels of the IL-17 family member IL-17Fin response to IL-23 treatment were measured by quantitative RT-PCR asin the legend to FIG. 2B.

[0019]FIG. 3: IL-23 acts on memory T cells to induce IL-17 productionMononuclear cells isolated from single cell suspension of murinesplenocytes were stained with (a) CyC-CD4+PE-CD44 or (b)CyC-CD4+PE-CD62L and sorted for CD4⁺ cells that were eitherCD44^(high)/CD62L^(low) for memory phenotype or CD44^(low)/CD62^(high)for naïve phenotype. The sorted cells were cultured with 100 U/MLrecombinant IL-2 in the presence or absence of IL-23 (or its boiled prepas a control), plate bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml)for 5 days, washed, and re-stimulated with anti-CD3 antibody for another24 hours. Supernatants were collected and IL-17 levels were measuredusing ELISA.

[0020]FIG. 4: IL12p40 antibody blocks IL-23-dependent IL-17 production:(A) Increasing concentrations of p40 antibody or an unrelatedisotype-matched control antibody were pre-incubated with IL-23 (100ng/ml) for 1 hr. at 37° C. and then incubated for another 5-6 days withmononuclear cells isolated from mouse spleen (2×10⁶ cells/ml) inpresence of recombinant IL-2. Supernatant were harvested and levels ofIL-17 measured using ELISA (left panel). Optimum concentrations ofIL-12p40 antibody or an unrelated isotype-matched control antibody werepre-incubated with conditioned media of LPS-stimulated dendritic cells(10% v/v) for 1 hr. at 37° C. and then incubated for another 5 days withmononuclear cells isolated from mouse spleen (2×10⁶ cells/ml) inpresence of recombinant IL-2. Supernatant were harvested and levels ofIL-17 measured using ELISA (right panel). (B) Mononuclear cells isolatedfrom splenocytes of wild type mice (C57/BL6) or mice lacking one of thecomponents of IL-12, i.e. IL12a^(−/−) (p35 knockout) or IL12b^(−/−) (p40knockout) were cultured in the presence of ConA for 3 days and IL-17levels measured in supernatants using ELISA.

[0021]FIG. 5: Effect of IL-12 on IL-17 production. (A) Mononuclear cellsisolated from spleen cell cultures were incubated in the presencepurified IL-23 (1 nM) and the indicated concentration of IL-12 for 5days and then washed and re-stimulated with ConA for another 24 hours.IL-17 levels were measured in cell supernatant using ELISA kits. (B):Mononuclear cells isolated from spleen cell cultures from wild type ormice lacking IL-12Rβ2 (IL-12Rβ2^(−/−) ko) were incubated in the presenceor absence of purified IL-23 (1 nM) for 5 days and then washed andre-stimulated with ConA for another 24 hours. IL-17 and IFN-γ levelswere measured in cell supernatant using ELISA kits.

[0022]FIG. 6: Targeting of the IL-23p19 locus. A: The native IL-23p19locus (top), the targeting construct (middle), and the correctlytargeted locus (bottom) are depicted to scale unless otherwise indicatedby double slashes. Open boxes indicate coding exons, and hatched boxesrepresent exons encoding 5′ and 3′ untranslated regions of the resultingmessenger RNA (mRNA). The four coding exons of the p19 gene arenumbered. Boxes with arrows indicate the promoter regions for neomycin(neo) and thymidine kinase (tk) selection cassettes, and an open boxlabeled EGFP indicates the location of an enhanced green fluorescentprotein reporter gene. Restriction sites used for cloning and analysisof the arms are labeled as follows: B, Bam HI; S, Sac II; E, Eco RI;,Bg, Bgl II; X, Xho I. The location of an antisense primer used toamplify the short arm is indicated by the letter P and an arrow. Thesize of restriction fragments resulting from digestion with Bam HI andEco RI are indicated in the wild type (WT) and the mutated (MUT) locus,and the locations of two probes used to detect these fragments bysouthern blot are shown by thick lines. B and C: Southern blot analysisof Bam HI digests probed with probe 1, and Eco RI digests probed withprobe II, respectively. DNA was extracted from wild-type (WT) embryonicstem (ES) cells, from ES clone 1c5, and from a wild type, a heterozygous(HET), and a knockout (KO) mouse. The identity of the band is indicatedat the left side of the blot, while its size is given on the right side.

[0023]FIG. 7: Total serum immunoglobulin levels IL-23p19^(−/−) mice.Serum levels of immunoglobulin isotypes were determined by isotypespecific ELISA from groups of 16 wild type (filled circles) andIL-23p19^(−/−) (open circles) mice. Immunoglobulin isotypes areindicated at the bottom of the graph.

[0024]FIG. 8: Humoral immune response in IL-23p19^(−/−) mice. A-G:Ovalbumin (OVA) specific levels of IgG1 (A), IgG2a (B), IgG2b (C), IgG3(D), IgE (E), and IgA (F) after one (1^(st)) and two (2^(nd))immunizations with OVA. Filled circles, wild-type mice; open circles,and IL-23p19^(−/−) mice, gray circles, and IL-12p40^(−/−) mice.Arbitrary units were calculated as described in methods and materials.The average of each group is indicated by both a black horizontal barand a numeric value at the bottom of the graph. Asterisks markstatistically significant P-values of less than 0.05.

[0025]FIG. 9: T-independent B-cell responses are normal inIL-23p19^(−/−) mice. Serum levels of TNP specific IgM was determined byELISA from mice immunized with TNP-LPS (type I, left) or TNP-Ficoll(type II, right). Filled circles, wild-type mice; open circles, andIL-23p19^(−/−) mice.

[0026]FIG. 10: Memory T-cell function. Wild type (filled circles) andIL-23p19^(−/−) mice (open circles) were immunized on day 0 withOvalbumin and challenged on day 21 with TNP-OVA. Serum was harvested ondays 0, 14, and 26 and tested by ELISA for the presence of TNP-specificIgG1 (A) and IgG2a (B). For IgG1, a commercially available standard wasused. For IgG2a, arbitrary units were calculated as described in methodsand materials.

[0027]FIG. 11: Delayed type hypersensitivity (DTH) reactions. Antigenspecific swelling is calculated as percent increase in footpad thicknessover the value measured just before the challenge. The results wereaveraged over all six mice in each group, and error bars represent thestandard deviations. A second wild-type group that was not sensitized isused as a control for swelling induced by the antigen alone. An asteriskinside a symbol indicates that the difference between the correspondinggroup and wild-type mice is statistically significant (P<0.05). WT, wildtype; p19ko, IL-23p19^(−/−) mice; p40ko, IL-12p40^(−/−) mice.

[0028]FIG. 12: Normal T-cell priming yet reduced levels of IL-17production by IL-23p19^(−/−) antigen presenting cells. A: in vitroallostimulation experiment of balb/c T-cells in combination withwild-type (black bars) or IL-23p19^(−/−) (white bars) dendritic cells.Naïve CD4⁺ T-cells and CD8⁻/CD11c⁺/MHC-II⁺ cells were isolated by FACSand incubated in the presence or absence of bacterial lipopeptides(BLP). Proliferation and cytokine levels in the supernatants weredetermined after a 5-day incubation period. APC, antigen presentingcells. B: In vivo T-cell response. Lymph node cell suspensions fromwild-type (black bars) or IL-23p19^(−/−) mice (white bars) immunizedwith KLH were isolated and restimulated in vitro with 25 μg/ml KLH.Proliferation and IL-17 levels were measured after 5 days in culture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0029] A. Definitions

[0030] Unless defined otherwise, technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. See, e.g. Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley& Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

[0031] The term “antagonist” is used herein in the broadest sense. AnIL-23 “antagonist” is a molecule, which partially or fully bocks,inhibits, neutralizes, prevents or interferes with a biological activityof IL-23, regardless of the underlying mechanism. For the purpose of thepresent invention, the biological activity preferably is the ability toinduce IL-17 production in activated T cells. Antagonists of IL-23 canbe identified, for example, based upon their ability to inhibit, block,or reverse IL-23 mediated IL-17 production in activated (e.g. memory) Tcell populations. For example a culture of activated T cells can beincubated with IL-23, in the presence and absence of a test compound,and IL-17 level monitored in the cell culture supernatant, e.g. byELISA. If the IL-17 level is lower in the presence of the test compoundthan in its absence, the test compound is an IL-23 antagonist.Alternatively, real-time RT-PCR can be used to monitor IL-17 mRNAexpression in a tissue also expressing IL-23, before and after treatmentwith a test compound. Decrease in IL-17 mRNA level in the presence ofthe test compound indicates that the compound is an IL-23 antagonist.Examples of IL-23 antagonists include, without limitation, neutralizingantibodies against a subunit, e.g. a p40 subunit, of a native sequenceIL-23 polypeptide, immunoadhesins comprising an IL-23 subunit fused toan immunoglobulin constant region sequence, small molecules, antisenseoligonucleotides capable of inhibiting translation and/or transcriptionof a gene encoding a subunit of a native sequence IL-23 polypeptide,decoys, e.g. genetic decoys of the IL-23 gene, etc. Similarly, IL-23antagonist include, without limitation, neutralizing antibodies againsta subunit, e.g. an IL-12Rβ1 or IL-23R subunit, of a native IL-23receptor, immunoadhesins comprising an IL-23 receptor subunit fused toan immunoglobulin constant region sequence, small molecules, antisenseoligonucleotides capable of inhibiting translation and/or transcriptionof a gene encoding a subunit of a native sequence IL-23 receptorpolypeptide, decoys, e.g. genetic decoys of an IL-23 receptor gene, etc.

[0032] The term “agonist” is used herein in the broadest sense. An IL-23agonist is any molecule that mimics a biological activity mediated by anative sequence IL-23, regardless of the underlying mechanism. For thepurpose of the present invention, the biological activity preferably isthe ability to induce IL-17 production in activated T cells. Examples ofIL-23 agonists include, without limitation, agonist antibodies against asubunit, e.g. an IL-12Rβ1 or IL-23R subunit, of a native IL-23 receptor,peptides and small organic molecules.

[0033] “Antisense oligodeoxynucleotides” or “antisense oligonucleotides”(which terms are used interchangeably) are defined as nucleic acidmolecules that can inhibit the transcription and/or translation oftarget genes in a sequence-specific manner. The term “antisense” refersto the fact that the nucleic acid is complementary to the coding(“sense”) genetic sequence of the target gene. Antisenseoligonucleotides hybridize in an antiparallel orientation to nascentmRNA through Watson-Crick base-pairing. By binding the target mRNAtemplate, antisense oligonucleotides block the successful translation ofthe encoded protein. The term specifically includes antisense agentscalled “ribozymes” that have been designed to induce catalytic cleavageof a target RNA by addition of a sequence that has natural self-splicingactivity (Warzocha and Wotowiec, “Antisense strategy: biological utilityand prospects in the treatment of hematological malignancies.” Leuk.Lymphoma 24:267-281 [1997]).

[0034] The term “antibody” is used in the broadest sense andspecifically covers monoclonal antibodies (including antagonist, e.g.neutralizing antibodies and agonist antibodies), polyclonal antibodies,multi-specific antibodies (e.g., bispecific antibodies), as well asantibody fragments. The monoclonal antibodies specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]). The monoclonalantibodies further include “humanized” antibodies or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv FR residuesof the human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andmaximize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); and Reichmann et al., Nature, 332:323-329 (1988).The humanized antibody includes a PRIMATIZED® antibody wherein theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.

[0035] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10):1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0036] As used herein, the term “inflammatory disease” or “inflammatorydisorder” refers to pathological states resulting in inflammation,typically caused by neutrophil chemotaxis. Examples of such disordersinclude inflammatory skin diseases including psoriasis and atopicdermatitis; systemic scleroderma and sclerosis; responses associatedwith inflammatory bowel disease (IBD) (such as Crohn's disease andulcerative colitis); ischemic reperfusion disorders including surgicaltissue reperfusion injury, myocardial ischemic conditions such asmyocardial infarction, cardiac arrest, reperfusion after cardiac surgeryand constriction after percutaneous transluminal coronary angioplasty,stroke, and abdominal aortic aneurysms; cerebral edema secondary tostroke; cranial trauma, hypovolemic shock; asphyxia; adult respiratorydistress syndrome; acute-lung injury; Behcet's Disease; dermatomyositis;polymyositis; multiple sclerosis (MS); dermatitis; meningitis;encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmunediseases such as rheumatoid arthritis (RA), Sjorgen's syndrome,vasculitis; diseases involving leukocyte diapedesis; central nervoussystem (CNS) inflammatory disorder, multiple organ injury syndromesecondary to septicaemia or trauma; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases includingglomerulonephritis; sepsis; sarcoidosis; immunopathologic responses totissue/organ transplantation; inflammations of the lung, includingpleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis,bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis,idiopathic pulmonary fibrosis (IPF), and cystic fibrosis; etc. Thepreferred indications include, without limitation, chronic inflammation,autoimmune diabetes, rheumatoid arthritis (RA), rheumatoid spondylitis,gouty arthritis and other arthritic conditions, multiple sclerosis (MS),asthma, systhemic lupus erythrematosus, adult respiratory distresssyndrome, Behcet's disease, psoriasis, chronic pulmonary inflammatorydisease, graft versus host reaction, Crohn's Disease, ulcerativecolitis, inflammatory bowel disease (IBD), Alzheimer's disease, andpyresis, along with any disease or disorder that relates to inflammationand related disorders.

[0037] The terms “treat” or “treatment” refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) an undesired physiological change ordisorder. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already with thecondition or disorder as well as those prone to have the condition ordisorder or those in which the condition or disorder is to be prevented.

[0038] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain thedesired effect for an extended period of time.

[0039] “Intermittent” administration is treatment that is notconsecutively done without interruption, but rather is cyclic in nature.

[0040] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0041] A “subject” is a vertebrate, preferably a mammal, more preferablya human.

[0042] The term “mammal” is used herein to refer to any animalclassified as a mammal, including, without limitation, humans, domesticand farm animals, and zoo, sports, or pet animals, such as sheep, dogs,horses, cats, cows, etc. Preferably, the mammal herein is human.

[0043] An “effective amount” is an amount sufficient to effectbeneficial or desired therapeutic (including preventative) results. Aneffective amount can be administered in one or more administrations.

[0044] B. Modes of Carrying Out the Invention

[0045] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, such as, “MolecularCloning: A Laboratory Manual”, 2^(nd) edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology”, 4^(th) edition (D.M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); “GeneTransfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds.,1994); and “Current Protocols in Immunology” (J. E. Coligan et al.,eds., 1991).

[0046] As discussed before, the invention is based on the recognitionthat IL-23 induces IL-17 production in activated T cell, in particularmemory cells, and that IL-23 antagonists are capable of inhibiting thisprocess. Accordingly, IL-23 antagonists are promising drug candidatesfor the treatment of inflammatory conditions characterized by elevatedlevels of IL-17. Conversely, IL-23 agonists are useful to induceprotective immune response to various infections, includingMycobacterial infections, such as, for example, Mycobacteriumtuberculosis (M. tuberculosis) infection.

[0047] 1. Screening Assays to Identify IL-23 Antagonists or Agonists

[0048] This invention includes screening assays to identify IL-23antagonists, which find utility in the treatment of inflammatoryconditions characterized by the presence of elevated levels of IL-17.The invention further includes screening assays to identify IL-23agonists that find utility in stimulating a protective immune responseto infections, such as infections by Mycobacterium tuberculosis.

[0049] Screening assays for antagonist drug candidates may be designedto identify compounds that bind or complex with IL-23 (including asubunit or other fragment thereof) or with an IL-23 receptor (includinga subunit or other fragment thereof), or otherwise interfere with theinteraction of IL-23 with other cellular proteins, thereby interferingwith the production or functioning of IL-23. The screening assaysprovided herein include assays amenable to high-throughput screening ofchemical libraries, making them particularly suitable for identifyingsmall molecule drug candidates. Generally, binding assays and activityassays are provided.

[0050] The assays can be performed in a variety of formats, including,without limitation, protein-protein binding assays, biochemicalscreening assays, immunoassays, and cell-based assays, which are wellcharacterized in the art.

[0051] All assays for antagonists and agonists are common in that theycall for contacting the drug candidate with an IL-23 polypeptide, or andIL-23 receptor polypeptide, or a fragment of such polypeptides(specifically including IL-23 and IL-23 receptor subunits) underconditions and for a time sufficient to allow these two components tointeract. For example, the human IL-23 p19 subunit is a 189 amino acidpolypeptide, the sequence of which is available from the EMBL databaseunder Accession Number AF301620 (NCBI 605580; GenBank AF301620; Oppmannet al., supra). The sequence of subunit p40 of the IL-23 polypeptide isalso known (also known as IL-12 p40 subunit; NCBI 161561). The sequenceof IL-12Rβ1, to which IL-23 binds, is available under Accession NumberNCBI 601604. The making of antibodies or small molecules binding to suchpolypeptides is well within the skill of the ordinary artisan.

[0052] In binding assays, the interaction is binding, and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, either the IL-23 or IL-23 receptor polypeptide orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the IL-23 or IL-23 receptor polypeptide and drying.Alternatively, an immobilized antibody, e.g., a monoclonal antibody,specific for the IL-23 polypeptide or the IL-23 receptor polypeptide tobe immobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

[0053] If the candidate compound is a polypeptide which interacts withbut does not bind to IL-23 or the IL-23 receptor, its interaction withthe respective polypeptide can be assayed by methods well known fordetecting protein-protein interactions. Such assays include traditionalapproaches, such as, e.g., cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers (Fieldsand Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, the other one functioning as the transcription-activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

[0054] Compounds that interfere with the interaction of IL-23 and otherintra- or extracellular components, in particular IL-17, can be testedas follows. Usually a reaction mixture is prepared containing IL-23 andthe intra- or extracellular component (e.g. IL-17) under conditions andfor a time allowing for the interaction of the two products. To test theability of a candidate compound to inhibit the interaction of IL-23 andIL-17, the reaction is run in the absence and in the presence of thetest compound. In addition, a placebo may be added to a third reactionmixture, to serve as positive control. Since IL-23 has been shown toinduce IL-17 production, the ability of the test compound to inhibit theIL-23/IL-17 interaction can, for example, be tested by measuring theamount of IL-17 in the absence and presence of the test compound. If theIL-17 amount is lower in the absence of the candidate compound than inits presence, the candidate compound is an IL-23 antagonist by thedefinition of the present invention.

[0055] The IL-23 antagonists identified based upon their ability toinhibit the induction of IL-17 production by IL-23 are drug candidatesfor the treatment of inflammatory conditions characterized by thepresence of elevated levels of IL-17.

[0056] The IL-23 agonists identified by upon their ability to promotethe induction of IL-17 production by IL-23 are drug candidates forevoking or supporting a protective immune response to infections, suchas infection by Mycobacterium tuberculosis, and, as a result, for thetreatment of infectious diseases, such as tuberculosis.

[0057] It is emphasized that the screening assays specifically discussedherein are for illustration only. A variety of other assays, which canbe selected depending on the type of the antagonist candidates screened(e.g. polypeptides, peptides, non-peptide small organic molecules,nucleic acid, etc.) are well know to those skilled in the art and areequally suitable for the purposes of the present invention.

[0058] 2. Anti-IL-23 and anti-IL-23 Receptor Antibodies

[0059] In a particular embodiment, the IL-23 antagonists or agonists aremonoclonal antibodies to IL-23 (e.g. a subunit of IL-23), includingantibody fragments. In another particular embodiment, the IL-23antagonists and agonists include monoclonal antibodies to an IL-23receptor (e.g. a subunit of an IL-23 receptor). IL-23, including itssubunits, has been discussed hereinabove. The receptor for IL-23 iscomprised of two subunits, IL-12Rβ1, and a more recently discoveredsubunit termed IL-23R (Parham et al., J. Immunol. 168:5699-5798 (2002)).Antibodies to either subunit are specifically within the scope of theinvention. In case of antagonists, antibodies specifically binding theIL-23R subunit are particularly preferred, since they specifically blockthe biological activities mediated by IL-23.

[0060] Methods for making monoclonal antibodies are well known in theart. Thus, monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

[0061] The immunizing agent will typically include the IL-23 or IL-23receptor polypeptide or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes (“PBLs”) are used if cells of human originare desired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

[0062] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0063] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst IL-23 or an IL-23 receptor. Preferably, the binding specificityof monoclonal antibodies produced by the hybridoma cells is determinedby immunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0064] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0065] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0066] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0067] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.In vitro methods are also suitable for preparing monovalent antibodies.

[0068] The anti-IL-23 and anti-IL-23 receptor antibodies of theinvention may further be humanized antibodies or human antibodies.Humanized forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

[0069] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0070] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0071] Mendez et al. (Nature Genetics 15: 146-156 (1997)) have furtherimproved the technology and have generated a line of transgenic micedesignated as “Xenomouse II” that, when challenged with an antigen,generates high affinity fully human antibodies. This was achieved bygerm-line integration of megabase human heavy chain and light chain lociinto mice with deletion into endogenous J_(H) segment as describedabove. The Xenomouse II harbors 1,020 kb of human heavy chain locuscontaining approximately 66 V_(H) genes, complete D_(H) and J_(H)regions and three different constant regions (μ, δ and χ), and alsoharbors 800 kb of human κ locus containing 32 Vκ genes, Jκ segments andCκ genes. The antibodies produced in these mice closely resemble thatseen in humans in all respects, including gene rearrangement, assembly,and repertoire. The human antibodies are preferentially expressed overendogenous antibodies due to deletion in endogenous J_(H) segment thatprevents gene rearrangement in the murine locus.

[0072] Alternatively, the phage display technology (McCafferty et al.,Nature 348, 552-553 (1990)) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol. 10, 779-783 [1992]). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This techniques allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires has been described by Waterhouse et al., Nucl. Acids Res.21, 2265-2266 (1993).

[0073] Various techniques have been developed for the production ofantibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al.,Science 229:81 (1985)). However, these fragments can now be produceddirectly by recombinant host cells. For example, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formF(ab′)₂ fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). Inanother embodiment, the F(ab′)₂ is formed using the leucine zipper GCN4to promote assembly of the F(ab′)₂ molecule. According to anotherapproach, Fv, Fab or F(ab′)₂ fragments can be isolated directly fromrecombinant host cell culture. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner.

[0074] Heteroconjugate antibodies, composed of two covalently joinedantibodies, are also within the scope of the present invention. Suchantibodies have, for example, been proposed to target immune systemcells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment ofHIV infection (PCT application publication Nos. WO 91/00360 and WO92/200373). Heteroconjugate antibodies may be made using any convenientcross-linking methods, using well known, commercially availablecross-linking agents.

[0075] For further information concerning the production of monoclonalantibodies see also Goding, J. W., Monoclonal Antibodies: Principles andPractice, 3rd Edition, Academic Press, Inc., London, San Diego, 1996;Liddell and Weeks: Antibody Technology: A Comprehensive Overview, BiosScientific Publishers: Oxford, UK, 1995; Breitling and Dubel:Recombinant Antibodies, John Wiley & Sons, New York, 1999; and PhageDisplay: A Laboratory Manual, Barbas et al., editors, Cold SpringsHarbor Laboratory, Cold Spring Harbor, 2001.

[0076] 3. Target Diseases

[0077] IL-17 has been implicated in various inflammatory diseases,including rheumatoid arhtritis (RA). One of the cardinal features of RAis erosion of periarticular bone. Osteoclasts play a key role in boneresorption but the mechanisms by which osteoclasts are formed fromprogenitor cells is not fully understood. Recently, Kotake, et al. (J.Clin. Invest. 103:1345 (1999)) reported that Interleukin 17 (IL-17)could induce the formation of osteoclast-like cells in cocultures ofmouse hemopoietic cells and primary osteoblasts. This IL-17 inducedosteoclastogenesis was shown to be inhibited by indomethacin, aselective inhibitor of cyclooxygenas-2 (COX-2). The synovial fluids fromRA patients were found to contain significantly higher levels of IL-17as compared to osteoarthritis (OA) patients. In addition, usingimmunostaining, IL-17-positive mononuclear cells were detected in thesynovial tissues of RA patients and not in tissue from OA patients.These findings have been interpreted to indicate that IL-17 maycontribute to bone erosion and joint damage in RA and may therefore, bea target for inhibition.

[0078] Behcet's disease patients have also been shown strinkinglyelevated serum levels of IL-17 compared to healthy subjects. Hamzaoui etal., Scand. J. Rheumatol. 31(4):205-10 (2002).

[0079] Elevated levels of IL-17 have been found within asthmaticairways, and it has been suggested that IL-17 might amplify inflammatoryresponses through the release of other proinflammatory mediators, suchas alpha-chemokines. Molet et al., J. Allergy Clin. Immunol.108(3):430-8 (2001); and Wong et al., Clin. Exp. Immunol. 125(2):177-83(2001).

[0080] Elevated levels of IL-17 have been reported for patients withsysthemic lupus erythrematosus. Wong et al., Lupus 9(8):589-93 (2000).

[0081] IL-17 has been described to play a role in psoriasis. Homey etal., J. Immunol. 164(12):6621-32 (2000).

[0082] It has been reported that IL-17 mRNA is augmented in blood andCSF mononuclear cells in multipe sclerosis. Matusevicius et al., Mult.Scler. 5(2): 101-4 (1999).

[0083] Based on these and numerous similar reports, IL-23 antagonists,which inhibit the ability of IL-23 to induce IL-17 production, andthereby lower IL-17 levels, are valuable candidates for the treatment ofa variety of (chronic) inflammatory conditions and diseases. Examples ofsuch conditions and diseases include, without limitation: chronicinflammation, autoimmune diabetes, rheumatoid arthritis (RA), rheumatoidspondylitis, gouty arthritis and other arthritic conditions, multiplesclerosis (MS), asthma, systhemic lupus erythrematosus, adultrespiratory distress syndrome, Behcet's disease, psoriasis, chronicpulmonary inflammatory disease, graft versus host reaction, Crohn'sDisease, ulcerative colitis, inflammatory bowel disease (IBD),Alzheimer's disease, and pyresis.

[0084] IL-17 is known to play an important role in the generation of aprotective response to certain infectious diseases, such as tuberculosisby promoting IFN-γ production and thereby inducing a cell-mediatedimmune response. Accordingly, IL-23 agonists, including agonistantibodies, find utility in inducing a cell-mediated immune response tovarious infections, such as tuberculosis causes by Mycobacteriumtuberculosis, and are promising drug candidates for treating thisinfectious disease which kills more than three million people worldwideevery year.

[0085] 4. Pharmaceutical Compositions

[0086] Antibodies specifically binding IL-23 or an IL-23 receptor, aswell as other IL-23 antagonist or agonist molecules identified by thescreening assays disclosed hereinbefore, can be administered for thetreatment of various disorders, in particular inflammatory diseases ordiseases benefiting from the induction of cell-mediated immune response,in the form of pharmaceutical compositions.

[0087] Where antibody fragments are used, the smallest inhibitoryfragment that specifically binds to the binding domain of the targetprotein is preferred. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993).

[0088] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

[0089] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0090] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0091] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Such molecules are suitably present in combination inamounts that are effective for the purpose intended, or may beformulated separately, and administered concurrently or consecutively,in any order.

[0092] For example, the IL-23 antagonists of the present invention maybe administered in combination with anti-inflammatory agents and otheractive compounds currently in use for the treatment of the targetdiseases and conditions. Such compounds include corticosteroids;non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin,ibuprofen, and COX-2 inhibitors, e.g. Celebrex® and Vioxx®;disease-modifying anti-rheumatic drugs (DMARDs), such as methotrexate,leflunomide, sulfasalazine, azathioprine, cyclosporine,hydroxychloroquine, and D-penicillamine; and biological responsemodifiers (BRMs), such as TNF and IL-1 inhibitors.

[0093] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0094] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLE 1

[0095] Interleukin-23 (IL-23) Promotes a Distinct CD4 T Cell ActivationState Characterized by the Production of Interleukin-17 (IL-17)

[0096] Although clearly produced by activated T cells, previous reportshave not provided clear classification of IL-17 within the paradigm ofTh1 and Th2 polarized cytokine profiles. The purpose of the initialexperiments described in this Example was to examine the possibilitythat IL-17 is expressed in response to signals distinct from thoseassociated with the Th1 or Th2 response.

[0097] Experimental Procedures

[0098] Cell Culture—Single cell suspensions of spleen were prepared fromC57/BL-6 mice, and mononuclear cells were isolated from suspendedsplenocytes by density gradient centrifugation. 2×10⁶ cells/ml werecultured with IL-2 (100 units/ml) in the presence or absence of variousstimuli (for times indicated in the figure legends), following which thecells were collected and analyzed for IL-17 using ELISA (R&D Systems,Minneapolis, Minn.). Dendritic cells were derived from macrophages(obtained as adherent population from splenocyte suspension) by treatingmactophages with rGM-CSF (2 ng/ml) and rIL-4 (1000 unites/ml) for 4days, washing and re-activating using LPS (0.5 μg/ml). Memory and naiveT cells were isolated by staining mononuclear cells isolated from singlecell suspension of murine splenocytes with CyC-CD4+PE-CD44 orCyC-CD4+PE-CD62L and sorting for CD4⁺ cells that were eitherCD44^(high)/CD62L^(low) for memory phenotype, or CD44^(low)/CD62^(high)for naive phenotype.

[0099] In vitro Induction of T Cell Differentiation —CD4⁺ cells wrepurified from spleen of wild type C57/BL6 mice using anti-CD4 magneticbeads (Miltenyi Biotech). Purified T cells (2×10⁶ cells/ml) wereactivated for 3 days by plating on plates coated with 5 μg/ml anti-CD3and 1 g/ml anti-CD28 antibodies. The cultures were supplemented withIL-2 and treated with IL-12 (20 mM)+anti-IL4 (0.5 μg/ml) (for Th1differentiation), or IL-23 (10 nM) (for IL-17 production). Followinginitial activation, the cell cultures were washed extensivly andre-stimulated with anti-CD3 (1 μg/ml) for another 24 h, following whichthe cell supernatants were analyzed for various secreted cytokines usingELISA.

[0100] IL-12p40 Antibody Inhibition of IL-17 Induction—Anti-IL-12antibody (R&D Systems, cat. no. AF-419-NA) or an unrelated controlantibody (anti-FGF-8b (R&D Systems, cat. no. AF-423-NA) werepre-incubated with IL-23 (100 ng/ml) or conditioned media ofLPS-stimulated dendritic cells (10% v/v) for 1 h at 37° C. and thenincubated for another 5-6 days with mononuclear cells isolated frommouse spleen (2×10⁶ cells/ml). Supernatants were collected and levels ofIL-17 measured using ELISA.

[0101] Purification of IL-23—Murine IL-23—IL-23 component was producedby co-expression of carboxyl-terminal His-tagged p19 and FLAG0tagged p40in human embryonic kidney cells (293 cells), and secreted protein waspurified by nickel affinity resin. Endotoxin levels were undetectable atless than 0.2 endotoxin units per μg.

[0102] Results

[0103] First, the ability of various microbial products to stimulate theproduction of IL-17 was examined. Increased IL-17 has recently beenobserved by Infante-Duarte et. al., J Immunol 165, 6107 (2000), inresponse to microbial lipopeptides from a Lyme disease causingspirochete, B. burgdorferi. Spleen cell cultures in the presence ofvarious microbial peptides including LPS (gram-negative bacteria), LTA(gram positive bacteria) or LBP (bacterial lipopeptide) resulted in theproduction of IL-17 (FIG. 1). Neither purified T cells alone, norpurified macrophages themselves produced IL-17. Purified T cells, uponreceptor cross-linking using plate-bound anti-CD3 and treatment withsupernatants from activated macrophages/dendritic cells producedincreased IL-17, indicating the presence of an unidentified factor(s)released by these cells that acts on T cells to promote IL-17production.

[0104] In profiling the expression of candidate molecules that might beresponsible for this IL-17 promoting activity, a 100-1000 fold increasedmRNA expression of the IL-23 (B. Oppmann et al., Immunity 13, 715(2000)) components p19 and p40 was observed in activated dendritic cellsusing real-time RT-PCR (not shown), hence, the effect of IL-23 wasexamined.

[0105] Murine IL-23 component was produced by co-expression of carboxylterminal His-tagged p19 and Flag-tagged p40 in human embryonic kidneycells (293 cells) and secreted protein was purified by nickel affinityresin. Endotoxin levels were undetectable at less than 0.2 EU per μg.Spleen cell cultures were incubated in presence of IL-2 (100U/ml) andConA (2.5 μg/ml) under Th1-inducing conditions (IL-12+ anti-IL-4),Th2-inducing conditions (IL-4+anti-IFN-γ), or purified IL-23 (100 ng/ml)for 3-4 days, following which, the cultures were washed andre-stimulated with ConA for another 24 hours. Levels of variouscytokines were measured using ELISA. The levels less than the lowestdilution of the standard curve range of ELISA kit were recorded as ‘notdetectable (N.D.)’. The results below are representative of threeexperiments performed independently.

[0106] Spleen cells, cultured under IL-12-stimulated Th1-inducingconditions resulted in marginal IL-17 production, whereas underTh2-inducing conditions there was no increased production of IL-17 overcontrols. The results are shown in the following Table 1. TABLE 1Control IL-12 IL-4 IL-23 IL-17 N.D 58 ± 82 64 ± 91 1191 ± 569  IL-4 50 ±26 396 ± 17  3259 ± 118  101 ± 100 IFN-γ 341 ± 0  2757 ± 1016 489 ± 502580 ± 813 GM-CSF N.D. 46 ± 13 365 ± 516 882 ± 169 TNF-α N.D 174 ± 40 214 ± 314 205 ± 85 

[0107] Presence of IL-23 in cultures resulted in high level IL-17production, in a dose-dependent manner (FIG. 2). IL-23 also resulted inhigher levels of GM-CSF than observed under Th1-inducing conditions. Incontrast, IFN-γ levels were significantly lower than those obtainedunder Th1-inducing conditions. TNF-α levels were similar to Th1conditions. IL-12p40 alone did not result in any IL-17 production (datanot shown). IL-23 promoted elevated levels of IL-17 mRNA (FIG. 2B).IL-17 mRNA levels were increased several hundred-fold within 6 h ofIL-23 exposure and remained elevated in the continued presence of IL-23.This effect was no inhibited by the presence of an antibody againstIL-17, suggesting that the IL-17 itself was not contributing to thisprocess (not shown). In addition, mRNA for IL-17F, a recently identifiedIL-17 family member, was also found to be upregulated in response toIL-23 (FIG. 2C).

[0108] IL-23 has been reported to promote the proliferation of memorybut not naïve T cells (D. M. Frucht, supra. Therefore, the effect ofIL-23 on IL-17 production from naïve versus memory T cell populationswas examined. Purified CD4⁺ T cells were isolated from splenocytes byfluorescence activated cell sorting (FACS). The memory cell populationwas selected as CD4⁺CD44^(high) (R. C. Budd et al., J Immunol 138, 3120(1987)), or CD4⁺CD62L^(low) (T. M. Jung, W. M. Gallatin, I. L. Weissman,M. O. Dailey, J Immunol 141, 4110 (1988)), and naïve cell population wasselected as CD4⁺CD44^(low) or CD4⁺CD62L^(high). As seen in FIG. 3, IL-23stimulated IL-17 production only in memory cell population (CD44^(high)and CD62L^(low)) and not in naïve cells (CD44^(low) or CD62L^(high)).

[0109] The IL-23-mediated IL-17 production was completely blocked in thepresence of a neutralizing IL-12 antibody that interacts with the p40subunit shared with IL-23 (FIG. 4A, left panel). This effect was not dueto ligation of Fc receptors on antigen presenting cells as there was nochange in IL-17 production in the presence of unrelated antibody. Thisantibody also inhibited >50 percent the induction of IL-17 productionobserved in response to conditioned media from LPS stimulated dendriticcells (FIG. 4A, right panel). A marked reduction, but not abrogation, ofIL-17 production was seen in response to ConA stimulation from spleencell cultures of mice lacking IL-12p40 component (strain:B6.129S1-IL12b^(tm1Jm)) as compared to wild type mice or mice lackingIL-12p35 component (strain: B6.129S1-IL12 a^(tm1Jm))(FIG. 4B).

[0110] In order to examine the role of IL-12 in IL-17 production,increasing amounts (0.001-1 nM) of murine IL-12 were added to IL-23 (1nM) containing cultures. As seen in FIG. 5A, IL-12 decreased IL-17levels in a dose dependent manner.

[0111] Additionally, splenocytes from mice lacking IL-12 receptor betachain 2 (IL-12Rβ2) (Wu et al., J Immunol 165, 6221 (2000)), the specificreceptor component of IL-12 (A. O. Chua, V. L. Wilkinson, D. H. Presky,U. Gubler, J Immunol 155, 4286 (1995)), were treated with purifiedIL-23. Splenocytes from IL-12Rβ2^(−/−) mice responded to IL-23 stimulusby increasing IL-17 production over the un-stimulated control (FIG. 5B)without affecting IFN-γ levels. Surprisingly, the background levels ofIL-17 in these mice were more than 10-fold as compared to wild-typemice, suggesting a possible negative regulation by IL-12 ofIL-23-induced IL-17 production. However, in contrast to IL-12Rβ2knockout mice, we did not observe increased IL-17 in spleen culturesfrom IL-12p35 knockout mice. The reasons for this difference are notknown, but could relate to alteration in IL-12p40 function in theabsence of p35, or differences in genetic background or pathogenexposure.

[0112] Discussion

[0113] Taken together, these data suggest a role for IL-23 in thepromotion of a distinct T cell activation state that expresses IL-17 asan effector cytokine. The Th1 and Th2 paradigms have been described aspromoting cell mediated versus humoral immune responses. These responsesprovide important defense for intracellular and extracellular pathogensrespectively, and defects in either of these responses are associatedwith increased susceptibility to specific pathogens. In contrast, IL-23may serve to promote an adaptive immune response to pathogens that ischaracterized by a heavy reliance on cells thought to function primarilyas mediators of the innate immune response. IL-17, as a principleeffector cytokine of this response, is able to promote the more rapidrecruitment of monocytes and neutrophils through induced chemokineproduction. In addition, the high level GM-CSF production observed inresponse to IL-23 supports the production of additional myeloid cells.This is further augmented by G-CSF production from localIL-17-stimulated stromal cells. The character of this adaptive responseis, however, not an exclusive reliance on phagocytic cells of themyeloid lineage response as IL-17 is known to promote the induction ofICAM by IL-17 thereby providing important co-stimulation of further Tcells responses.

[0114] Recently, several studies have pointed out significantdifferences between mice deficient in p35 and mice deficient in p40(Decken et al., Infect Immun. 66:4994-5000 (2002); Cooper et al., J.Immunol. 168:1322-1327 (2002); Elkins et al., Infection & Immunity70:1936-1948; Holscher et al., J. Immunol. 167:6957-6966 (2001)). Thesestudies share the observation that loss of p40 is generally moredeleterious than loss of p35 in the immune-mediated clearance of avariety of model organisms.

[0115] The association of IL-17 expression with a number of seriousinflammatory diseases suggests that IL-23 antagonists may be promisingdrug candidates in the treatment of such diseases.

EXAMPLE 2

[0116] Interleukin-23 (IL-23) Deficient Mice

[0117] To further investigate the relationship between IL-23 and IL-17in vivo, the phenotype of IL-23 deficient mice was compared to that ofIL-17 deficient animals.

[0118] Experimental Procedures

[0119] Mice: All mice were housed under specific pathogen freeconditions. IL-12p40^(−/−) mice were obtained from the Jacksonlaboratory (Bar Harbor, Mass.), and C57BL/6 were obtained from CharlesRiver laboratories (San Diego, Calif.).

[0120] Reagents: Unless otherwise indicated, reagents were purchasedfrom the following suppliers: Antibodies and ELISA reagents wereobtained from BD Pharmingen (San Diego, Calif.), cytokines from R&Dsystems (Minneapolis, Minn.), TNP-coupled antigens from BiosearchTechnologies (Novato, Calif.) and tissue culture reagents fromInvitrogen (Carlsbad, Calif.).

[0121] Generation of IL23p19 deficient mice. Genomic DNA encompassingthe murine IL23p19 locus was isolated from clone 198a3 of a genomic BAClibrary by Genome Systems (Incyte Genomics, Palo Alto, Calif.). Atargeting vector designed to replace the entire IL23p19 coding regionwith an EGFP reporter gene was constructed from the following DNAfragments using standard molecular cloning techniques: a thymidinekinase selection cassette; a 5′ homology arm of 5403 base pairs definedby endogenous SacII and BglII sites on the distal and proximal ends,respectively; an EGFP expression cassette excised from pEGFP-1 (BDClontech, Palo Alto, Calif.) using BamHI (5′-end) and AflIII (3′-end); aPGK-neo resistance cassette; and a 1203 bp short arm defined by anendogenous XhoI site at the proximal end and the primer5′-GCTTGGTGGCCCACCTATGAT-3′ (SEQ ID NO: 1) at the distal end (FIG. 6A).This construct was electroporated into 129/SvEv embryonic stem (ES)cells (Huang et al., Science 259:1742 (1993)) and homologousrecombination occurred in 9 out of 600 clones following selection withG418 and Gancyclovir. To verify correct targeting of the locus, genomicDNA from ES cells and animals was analyzed by southern blot. Digestionwith BaniHI followed by hybridization of membranes with probe 1 (a 831bp genomic DNA fragment obtained by PCR with oligos5′-AGACCCTCAAAGTTCATGAC-3′ (sense) (SEQ ID NO: 2) and5′-CTGACGGCGCTTTCTCTACC-3′ (antisense) (SEQ ID NO: 3)) yielded a 7027 bpfragment for the wild-type allele and an 11788 bp fragment for thecorrectly targeted mutant allele. Similarly, digestion of genomic DNAwith EcoRI followed by hybridization of membranes with probe 2 (a 390 bpgenomic DNA fragment obtained by PCR with oligos5′-TTTTGCCAGTGGGATACACC-3′ (sense) (SEQ ID NO: 4) and5′-AACTGCTGGGGCTGTTACAC-3′ (antisense) (SEQ ID NO: 5)) yielded a 9197 bpfragment for the wild-type allele and an 6211 bp fragment for thecorrectly targeted mutant allele. Two ES cell clones (1c5 and 3h6) wereinjected into blastocysts, and chimeric animals that transmitted themutant allele in their germline were obtained. For routine genotyping,we used a PCR-based method with a common antisense primer(5′-GCCTGGGCTCACTTTTTCTG-3′) (SEQ ID NO: 6), and wild-type specific(5′-GCGTGAAGGGCAAGGACACC-3′) (SEQ ID NO: 7) and knockout-specific(5′-AGGGGGAGGATTGGGAAGAC-3′ (SEQ ID NO: 8)) sense primers. Thisprimer-triplet amplifies a 210 bp fragment for the wild-type allele anda 289 bp fragment for the mutant allele. PCR was carried out in aRobocycler (Stratagene, La Jolla, Calif.), using the followingconditions: 1 cycle of 94° C., 60″; 35 cycles of 94° C., 30″, 58° C.,30″, 72° C., 60″; 1 cycle of 72° C., 7″.

[0122] FACS analysis of blood cell subsets: Spleens, thymi, and lymphnodes were isolated from 6-8 week old mice, and single cell suspensionswere prepared by standard methods. Peripheral blood was obtained bycardiac puncture and treated with EDTA to prevent coagulation, anderythrocytes were lysed using ACK lysing buffer (Biosource, Camarillo,Calif.). All cells were incubated for 30 minutes on ice in Hanksbalanced salt solution (HBSS) supplemented with 2% heat inactivatedbovine calf serum. Cells were then stained in the same buffer with 1 μgper million cells of various antibodies coupled to phycoerythrin, biotinor Cychrome™. Where biotinylated antibodies were used,streptavidin-coupled PE-TR conjugate (Caltag, Burlingame, Calif.) wasused for detection. After two washes with the same buffer, fluorescencewas detected using an Epics-XL flow cytometry system (Beckman CoulterInc., Fullerton, Calif.).

[0123] Stimulation of allotypic T-cells: CD4 and CD62L double positiveT-cells were isolated from the spleens of 6-8 week old balb/c mice by atwo-step isolation protocol. First, T-cells were depleted of other celltypes by a negative magnetic selection (Miltenyi, Auburn, Calif.). Thesecells were then labeled with antibodies against CD4 and CD62L and sortedby FACS on a MoFlo sorter (DakoCytomation, Fort Collins, Colo.).Dendritic cells from wild type or IL-23p19^(−/−) mice, both in theC57BL/6 background, were also isolated by a two-step protocol. CD11cpositive splenocytes were positive selected by magnetic separation(Miltenyi, Auburn, Calif.) prior to labeling with antibodies againstCD11c, MHC class II, and CD8. CD11c⁺/MHC-II⁺/CD8⁻ cells were then sortedby FACS, again using a MoFlo sorter. All populations used in theexperiment were at least 98% pure. To elicit allostimulatory responses,10⁴ dendritic cells and 10⁵ T-cells were incubated in a total of 200 μlof IMDM supplemented with penicillin-streptomycin and 10% heatinactivated bovine calf serum (Hyclone, Logan, Utah) in duplicates. Insome cases, 100 ng/ml bacterial lipopeptides was added to stimulatecytokine production by dendritic cells. After 5 days of incubation, 120μl of supernatant were removed for cytokine measurement by ELISA, andreplaced with fresh medium containing 1 μCi ³H-thymidine per well.Thymidine incorporation was determined 16 hours later using a Top Countliquid scintillation counter according to the manufacturers instructions(Packard Instruments, Meriden, Conn.).

[0124] In vivo T-cell differentiation: Four male and four female miceper group were immunized into the left hind footpad with 75 μg ofkeyhole limpet hemocyanin (KLH) (Sigma, St. Louis, Mo.) in 30 μl of a1:1 emulsion of CFA (BD Biosciences, San Diego, Calif.) and PBS.Draining inguinal and popliteal lymph nodes were harvested 5 days laterand restimulated in IMDM supplemented with penicillin-streptomycin, 10%heat inactivated bovine calf serum (Hyclone, Logan, Utah), and 25 μg/mlKLH. For proliferation assay, 5*10⁵ cells were seeded in 200 μl intriplicates in 96 well plates and allowed to proliferate for 112 hourswith addition of 1 μCi ³H-thymidine per well during the last 18 hours ofthe incubation period. Thymidine incorporation was determined using aTop Count liquid scintillation counter according to the manufacturersinstructions (Packard Instruments, Meriden, Conn.). For cytokinesecretion, 2.5*10⁶ cells were incubated in 1 ml in 48 well plates, andsupernatants were harvested after 72 hours. Cytokine secretion wasdetermined by ELISA. The data presented is one representative out ofthree total experiments.

[0125] Delayed type hypersensitivity responses: 6 mice per group weresubcutaneously injected with 200 μg of methylated bovine serum albumin(mBSA) (Sigma, St. Louis, Mo.) at three sites in the abdomen in acombined total of 200 μl of a 1:1 emulsion of CFA (BD Biosciences, SanDiego, Calif.) and PBS. On day 8 following immunization, the mice werechallenged by injection of 20 μl of 5 mg/ml mBSA in PBS into one rearfootpad, while the other rear footpad received 20 μl of PBS.Measurements of footpad swelling were taken at 18, 42, 66 hours afterchallenge, using a series 7 spring-loaded caliper (Mitutoyo, City ofIndustry, Calif.). The magnitude of the DTH responses was determinedfrom differences in footpad thickness between the antigen- andPBS-injected footpads.

[0126] T-dependent humoral responses and immunoglobulin analysis: Forthe measurement of total immunoglobulin levels, serum was obtained from8 male and 8 female, 6-9 week old, unimmunized mice of either genotype.Total immunoglobulin isotype levels were measured by Luminex bead assay(Upstate, Lake Placid, N.Y.). To assess the OVA specific humoral immuneresponse, groups of 7 mice per genotype (4 males and 3 females) wereimmunized with OVA in CFA on day 0 and received booster immunizations ofthe same antigen in incomplete Freund's adjuvant (IFA) (Sigma, St.Louis, Mo.) on days 21 and 42. For serum analysis, blood was obtained byretro-orbital bleeding before immunization and on days 14, 28, and 49after immunization. OVA specific immunoglobulin isotypes were detectedby ELISA, using OVA as a capture agent and isotype specific secondaryantibodies for detection. In order to be in the linear range of theELISA, serum samples were diluted as follows: 1:3125000 for IgG1,1:25000 for IgG2a, 1:625000 for IgG2b, and 1:1000 for IgG3, IgM, IgA andIgE. A dilution series of a serum obtained from an OVA-immunized mousefrom a previous experiment was used as a standard, since purified, OVAspecific isotypes are not commercially available. Results are expressedas arbitrary units, where the average of the wild-type group in the lastbleed was set as 100. To assess the contribution of memory T-cells tothe humoral response, groups of 5-6 mice of either genotype wereimmunized with OVA in CFA on day 0 and received a booster immunizationof TNP₁₁-OVA in IFA on day 21. For serum analysis, blood was obtained byretro-orbital bleeding before immunization and on days 14 and 28 afterimmunization. TNP specific immunoglobulin isotypes were detected byELISA, using TNP₂₈-BSA as a capture agent and isotype specific secondaryantibodies for detection. For TNP-specific IgG1, a commerciallyavailable standard was used. For TNP-specific IgG2a, a dilution seriesof a serum obtained from a TNP immunized mouse from a previousexperiment was used, and results were calculated as described above. Thesample dilutions were 1:31250 for IgG1 and 1:1250 for IgG2a.

[0127] T-independent humoral responses: Groups of 6 mice per genotypewere immunized intraperitoneally with 50 μg TNP₁-LPS or 100 μgTNP₂₀-AECM-Ficoll in PBS. Serum was harvested 10 days later, andTNP-specific IgM was analyzed by ELISA, using TNP₂₈-BSA as a captureagent and an IgM specific secondary antibody for detection. A TNPspecific IgM antibody was used as a standard for the ELISA. The sampledilutions were 1:1280 for Ficoll and 1:5120 for LPS.

[0128] Results

[0129] Deletion of the IL-23p19 gene. To determine the non-redundant invivo effects of IL-23, mice were generated that are deficient in IL-23but competent to produce IL-12. A targeting vector was constructed inwhich the entire coding region of p19, consisting of 4 exons, isreplaced by an enhanced GFP (eGFP) reporter gene, and a neomycinresistance cassette (FIG. 6). Germline transmission was obtained fromtwo correctly targeted ES cell clones, 1c5 and 3h6, and the mutation wasbackcrossed into the C57BL/6 background using speed congenics with 3markers per chromosome. Based on this analysis, only those mice wereselected in which the genetic contamination from the 129 background wasless than 5% for experiments. The pattern of eGFP expression wascomparable to that of endogenous p19 mRNA (data not shown).

[0130] IL-23p19^(−/−) mice have no overt phenotype. As expected from thephenotype of IL-23/IL-12 double deficient IL-12p40^(−/−) mice,IL-23p19^(−/−) animals did not display any overt phenotype and were bornat mendelian frequencies. No abnormalities in organs were found uponhistopathological examination, and further analysis of clinicalchemistry and hematology parameters did not reveal differences betweenwild type and knockout animals. Furthermore, IL-23p19^(−/−) mice werenormal in size and weight, and both sexes were fully fertile. Flowcytometric analysis of thymocytes, splenocytes, and peripheral bloodleukocytes with various cell surface markers did not indicate any majordifferences between wild-type and IL23p19^(−/−) animals (Table 2).Because IL-23 is known to act on memory T-cells, we the ratio of memory(CD44^(high)CD62L⁻) versus naïve (CD62L⁺) cells of each subset wasdetermined, but no difference was found between wild-type andIL-23p19^(−/−) mice. In the entire analysis, the only noticeabledifference between the two genotypes consists in a slight skewing of thedendritic cell subpopulations towards a CD8⁺ phenotype. While the effectwas minor, it reached statistical significance due to the tightness ofthe data, and could be compatible with recent observations that IL-23has effects on antigen presenting cells. In summary, IL-23 does notappear to be required for normal development, and the introduction of aneGFP cassette does not have a toxic effect on any cell type tested.TABLE 2 wild-type knockout P (diff.) Thymus CD4+  5.7 +/− 0.5  5.5 +/−0.0 0.504 CD8+  3.3 +/− 0.1  3.1 +/− 0.3 0.397 DN 25.0 +/− 4.2 17.0 +/−8.0 0.202 DP 65.9 +/− 3.7 74.3 +/− 8.0 0.174 Spleen CD4+ 24.3 +/− 0.822.5 +/− 2.7 0.342 % naive 69.0 +/− 1.3 67.5 +/− 2.1 0.090 % memory 29.1+/− 1.2 31.0 +/− 1.9 0.029 CD8+ 15.2 +/− 1.2 12.3 +/− 2.0 0.101 % naive64.1 +/− 5.4 67.0 +/− 2.8 0.199 % memory 18.1 +/− 1.8 18.3 +/− 1.4 0.084I-A(b)+/CD11c+  2.0 +/− 0.2  2.2 +/− 0.2 0.041 % CD8+ 12.8 +/− 0.9 16.3+/− 1.7 0.000 % CD8− 87.2 +/− 0.9 83.6 +/− 1.8 0.000 CD19+ 52.4 +/− 2.055.2 +/− 6.5 0.512 B220+ 52.0 +/− 2.0 55.5 +/− 5.3 0.360 NK1.1+  3.2 +/−0.1  2.8 +/− 0.1 0.055 Peripheral blood CD3+ 47.9 +/− 2.6 44.9 +/− 3.60.053 CD4+ 28.2 +/− 2.3 26.9 +/− 2.5 0.270 CD8+ 16.5 +/− 0.8 15.6 +/−1.7 0.150 CD19+ 43.2 +/− 3.2 45.2 +/− 3.6 0.215 B220+ 44.9 +/− 3.5 46.4+/− 4.8 0.466 DX5+  9.9 +/− 3.0  9.7 +/− 5.0 0.929 CD16+  8.0 +/− 0.9 8.6 +/− 1.5 0.302 I-A(b)+ 44.0 +/− 1.9 45.4 +/− 4.9 0.428

[0131] Humoral immune responses in IL-23p19^(−/−) mice. To determine therole of IL-23 in the generation of a humoral immune response, firsttotal immunoglobulin levels of all isotypes were measured in serum of 16mice of either genotype. There was no statistically significantdifference between wild type and IL-23p19^(−/−) mice (FIG. 7),indicating that the IL-23 is not critically required for the maintenanceof normal immunoglobulin levels. Next, we tested whether IL-23 isinvolved in the generation of a T-dependent humoral response against aprotein antigen delivered in adjuvant. To this end, groups of 7 micewere immunized, each with Ovalbumin (OVA), and assessed OVA-specificimmunoglobulin isotypes in preserum (all negative, data not shown), andafter each of two consecutive immunizations (FIG. 8). After primaryimmunization, none of the groups differed from each other significantlyfor OVA specific IgG1, IgG2b, IgG3, and IgE. However, significantlyreduced levels of OVA specific IgG2a and IgA in IL-23p19^(−/−) andIL-12p40^(−/−) animals were observed after primary immunization. Asexpected, the levels of all isotypes were increased dramatically afterthe second immunization. At this point, both IL-23p19^(−/−) andIL-12p40^(−/−) mice displayed marked reduction of all isotypes tested.The difference between these two genotypes was generally notsignificant, indicating that endogenous IL-12 does not play a major rolein the humoral response in the absence of IL-23.

[0132] Because humoral immune responses depend of the proper function ofboth B and T cells, we next sought to determine by what mechanism IL-23exerts its stimulatory effects. To test whether B-cell function isdirectly affected by the lack of IL-23, we tested the ability of IL-23deficient mice to mount B-cell responses against T-independent (TI)antigens. The TI-1 antigen trinitrophenyl- (TNP-) LPS leads to B-cellactivation via CD14 and TLR4, while the TI-2 antigen TNP-Ficollactivates B-cells through clustering of surface B-cell receptors.IL-23p19^(−/−) mice mounted normal B-cell responses to both types ofantigens (FIG. 9), indicating that IL-23 does not play a role inT-independent B-cell responses. Furthermore, B-cells from IL-23p19^(−/−)mice proliferated normally in vitro in response to LPS, anti-IgM, andanti-CD40 and underwent normal isotype switching in response to IL-4(not shown). IL-23 stimulation of B-cells did not lead to increasedproliferation or isotype switching (not shown), and thus we concludethat IL-23 does not directly affect B-cell function.

[0133] Because the humoral immune response was mainly compromised at thestage of the secondary immunization, and because B-cell functionappeared normal in IL-23p19^(−/−) mice, we hypothesized that inefficientre-activation of antigen specific helper T-cells might cause thephenotype. To address this question more directly, we immunized groupsof 5-6 mice with OVA on day 0, followed by a secondary immunization withTNP conjugated OVA on day 14. By using this immunization regimen, memoryT-cells specific for OVA are re-activated by the secondary immunization,but a novel set of B-cells with specificity for TNP is activated at thesecondary time point only. Therefore, the OVA specific memory B-cellsubset does not contribute to the formation of TNP-specificimmunoglobulins. Seven days after the booster, we tested for TNPspecific IgG1 and IgG2a in the serum, and found both isotypes to besignificantly reduced in IL-23p19^(−/−) mice (FIGS. 10A, B). This resultfurther underlines the importance of IL-23 in T-dependent B-cellresponses.

[0134] Delayed type hypersensitivity (DTH) responses in IL-23p19^(−/−)mice. To further investigate the function of memory CD4⁺ cells in IL-23p19^(−/−) mice, the ability of these animals to mount DTH responses wasevaluated. DTH responses are strongly T-cell dependent and were reportedto be defective in IL-12p40^(−/−) mice, but appear to be normal in micelacking IL-12p35, suggesting that they might be mediated by IL-23. Toaddress this question, we sensitized groups of 6 wild-type,IL-23p19^(−/−), and IL-12p40^(−/−) animals each with methylated BSA(mBSA) in complete Freund's adjuvant (CFA) and elicited DTH responses 7days later by injection of MBSA into footpads. To control fornonspecific swelling, we also challenged a group of wild-type mice thathad not been sensitized. Specific footpad swelling was measured 18, 42,and 66 hours after the challenge and found to be inhibited to a similardegree in both IL-12p40^(−/−) and IL-23p19^(−/−) mice compared towild-type mice (FIG. 11). The kinetics was also similar, with bothIL-12p40^(−/−) and IL-23p19^(−/−) mice showing strongly reduced swellingat the 42 and 66 but not at the 18 hour time point. Therefore, IL-23 isa principal mediator of DTH responses, and lack of IL-23 leads toinefficient responses by memory CD4⁺ T-cells.

[0135] Capacity of IL-23p19^(−/−) dendritic cells to stimulate T-cells.To rule out the possibility that the defects observed in IL-23p19^(−/−)mice are due to inefficient T-cell priming by IL-23 deficient antigenpresenting cells, we next investigated the potential of IL-23p19^(−/−)DC to stimulate allotypic naïve CD4⁺ T-cells isolated from the spleensof balb/c mice. In the absence of DC, these T-cells did not proliferatenor secrete appreciable amounts of cytokines (FIG. 12A). Addition of DCof either genotype resulted in robust proliferation and production ofIL-2 in both genotypes. Since we have shown previously that IL-23 is apotent inducer of IL-17, we next induced IL-23 production by DC usingbacterial lipopeptides, a potent Toll-like receptor- (TLR-) 2 agonistand inducer of IL-23 production. Under these conditions, wt DC potentlyinduced IL-17 production by the T-cells (FIG. 12A, bottom panel), whileT-cells stimulated with IL-23p19^(−/−) DC produced significantly lessIL-17. To confirm these observations in a more physiological setting, wenext elicited T-cell responses in vivo by immunizing groups of 8 micewith Keyhole-limpet hemocyanin (KLH) in complete Freund's adjuvant(CFA). Draining lymph node cells (LNC) were harvested 5 days later andre-stimulated with KLH in vitro. Again, we observed that LNC harvestedfrom IL-23p19^(−/−) mice produced significantly less IL-17 (FIG. 12B,bottom panel). LNC proliferation was comparable in both genotypes (FIG.12B, top panel), indicating that both wt and IL-23p19^(−/−) mice mountedrobust T-cell responses against the antigen. Thus, IL-23 deficiency doesnot grossly impair the stimulatory potential of dendritic cells, butresults in attenuated IL-17 production by T-cells.

[0136] Discussion

[0137] Using IL-23p19 deficient mice, the non-redundant in vivofunctions of IL-23 were assessed, and found that IL-23 deficiencyresults in compromised T-cell dependent immune responses, such ashumoral immune responses and DTH reactions.

[0138] Profoundly reduced humoral immune responses were observed inIL-23p19^(−/−) mice, affecting all immunoglobulin isotypes. In parallel,responses of IL-12p40^(−/−) mice were inhibited to a similar or slightlyhigher degree. Our results support the conclusion that IL-23 isabsolutely required for an efficient humoral response, while it remainsto be determined, through the use of IL-12p35^(−/−) mice, whether IL-23is sufficient for normal humoral responses in the absence of IL-12.

[0139] In summary, IL23p19^(−/−) mice have attenuated in vivo T-cellresponses manifesting in reduced DTH and humoral immune responses, andphenotypically resemble IL-17 deficient mice. Our results indicate thatclinical administration of IL-23 or its agonists might be beneficial tosupport T-cell function in immunization regimens and inimmunocompromised patients.

[0140] While the present invention has been described with reference towhat are considered to be the specific embodiments, it is to beunderstood that the invention is not limited to such embodiments. To thecontrary, the invention is intended to cover various modifications andequivalents included within the spirit and scope of the appended claims.

1 8 1 21 DNA Artificial Sequence Primer 1 gcttggtggc ccacctatga t 21 220 DNA Artificial Sequence Sense primer 2 agaccctcaa agttcatgac 20 3 20DNA Artificial Sequence Antisense primer 3 ctgacggcgc tttctctacc 20 4 20DNA Artificial Sequence Sense primer 4 ttttgccagt gggatacacc 20 5 20 DNAArtificial Sequence Antisense primer 5 aactgctggg gctgttacac 20 6 20 DNAArtificial Sequence Common antisense primer 6 gcctgggctc actttttctg 20 720 DNA Artificial Sequence Wild-specific specific sense primer 7gcgtgaaggg caaggacacc 20 8 20 DNA Artificial Sequence Knockout specificsense primer 8 agggggagga ttgggaagac 20

What is claimed is:
 1. A method for inhibiting interleukin-17 (IL-17)production by T cells comprising treating said T cells with anantagonist of interleukin-23 (IL-23).
 2. The method of claim 1 whereinsaid T cells are activated T cells.
 3. The method of claim 1 whereinsaid T cells are memory cells.
 4. The method of claim 1 wherein saidtreatment is performed in vivo.
 5. The method of claim 1 wherein saidtreatment is performed in a mammalian subject.
 6. The method of claim 5wherein said mammalian subject is human.
 7. The method of claim 6wherein said antagonist is an anti-IL-23 or an anti-IL-23 receptorantibody.
 8. The method of claim 7 wherein said antibody is an antibodyfragment.
 9. The method of claim 8 wherein said antibody fragment isselected from the group consisting of Fv, Fab, Fab′, and F(ab′)₂. 10.The method of claim 7 wherein said antibody is a full-length antibody.11. The method of claim 7 wherein said antibody is chimeric.
 12. Themethod of claim 7 wherein said antibody is humanized.
 13. The method ofclaim 7 wherein said antibody is human.
 14. A method for the treatmentof an inflammatory disease characterized by elevated expression ofinterleukin 17 (IL-17) in a mammalian subject, comprising administeringto said subject an effective amount of an antagonist of interleukin-23(IL-23).
 15. The method of claim 14 wherein said mammalian subject ishuman.
 16. The method of claim 15 wherein said inflammatory disease isselected from chronic inflammation, autoimmune diabetes, rheumatoidarthritis (RA), rheumatoid spondylitis, gouty arthritis and otherarthritic conditions, multiple sclerosis (MS), asthma, systhemic lupuserythrematosus, adult respiratory distress syndrome, Behcet's disease,psoriasis, chronic pulmonary inflammatory disease, graft versus hostreaction, Crohn's Disease, ulcerative colitis, inflammatory boweldisease (IBD), Alzheimer's disease, and pyresis.
 17. The method of claim16 wherein said inflammatory disease is a chronic inflammatory disease.18. The method of claim 17 wherein said chronic inflammatory disease isselected from the group consisting of rheumatoid arthritis (RA), graftversus host reaction, multiple sclerosis (MS), and psoriasis.
 19. Themethod of claim 15 wherein said antagonist is an anti-IL-23 or ananti-IL-23 receptor antibody.
 20. The method of claim 19 wherein saidantibody is an antibody fragment.
 21. The method of claim 20 whereinsaid antibody fragment is selected from the group consisting of Fv, Fab,Fab′, and F(ab′)₂.
 22. The method of claim 19 wherein said antibody is afull-length antibody.
 23. The method of claim 19 wherein said antibodyis chimeric.
 24. The method of claim 19 wherein said antibody ishumanized.
 25. The method of claim 19 wherein said antibody is human.26. The method of claim 15 wherein said antagonist is administered incombination with an additional therapeutic agent.
 27. The method ofclaim 26 wherein said additional therapeutic agent is ananti-inflammatory molecule.
 28. The method of claim 27 wherein saidanti-inflammatory molecule is selected from the group consisting ofcorticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs). 29.A method for identifying an anti-inflammatory agent comprising the stepsof: (a) incubating a culture of T cells with IL-23, in the presence andabsence of a candidate molecule; (b) monitoring the level of IL-17 insaid culture; and (c) identifying said candidate molecule as ananti-inflammatory agent if the level of IL-17 is lower in the presencethan in the absence of said candidate molecule.
 30. The method of claim29 wherein said candidate molecule is a non-peptide small organicmolecule.
 31. The method of claim 29 wherein said candidate molecule isa peptide.
 32. The method of claim 29 wherein said candidate molecule isa polypeptide.
 33. The method of claim 29 wherein said candidatemolecule is an antibody.
 34. The method of claim 29 wherein said T cellsare activated T cells.
 35. The method of claim 29 wherein said T cellsare memory cells.
 36. The method of claim 29 wherein the level of IL-17is monitored by ELISA.
 37. An anti-inflammatory agent identified by themethod of claim
 29. 38. A method for inducing IL-17 production in amammalian subject comprising administering to said subject an IL-23agonist.
 39. The method of claim 38 wherein said mammalian subject ishuman.
 40. The method of claim 39 wherein the human subject has beenexposed to bacterial infection.
 41. The method of claim 40 wherein thehuman subject has been exposed to infection by Mycobacteriumtuberculosis.
 42. The method of claim 39 wherein said IL-23 agonist isan antibody.
 43. The method of claim 42 wherein said antibody is ananti-IL-23 or anti-IL-23 receptor antibody.
 44. The method of claim 43wherein said antibody is an antibody fragment.
 45. The method of claim44 wherein said antibody fragment is selected from the group consistingof Fv, Fab, Fab′ and F(ab′)₂.
 46. The method of claim 43 wherein saidantibody is a full-length antibody.
 47. The method of claim 43 whereinsaid antibody is chimeric.
 48. The method of claim 43 wherein saidantibody is humanized.
 49. The method of claim 43 wherein said antibodyis human.